This Phase II STTR proposal describes a collaborative effort between researchers in the Department of Pharmaceutical Sciences at the University of Connecticut and Promiliad Biopharma to develop efficacious antibiotics targeting both methicillin-resistant strains of Staphylococcus aureus (MRSA) and Streptococcus pyogenes. We have recently completed a Phase I STTR grant that was focused on developing agents against the parasitic protozoan Cryptosporidium hominis by targeting the essential enzyme dihydrofolate reductase (DHFR). Through this work we developed the most potent and selective inhibitors of this enzyme reported to date and showed efficacy against the cultured parasite. In parallel with these efforts targeting Cryptosporidium, we examined the generality of this compound class to inhibit DHFR from other pathogenic organisms and found that this scaffold can be customized to potently target a wide range of pathogenic DHFR enzymes, including that from MRSA and Streptococcus pyogenes. We intend to capitalize on the high-profile nature of these organisms by switching the focus of our first proof-of-concept antibiotic. We have shown that the compounds effectively inhibit the growth of various phenotypes of MRSA and Streptococcus pyogenes. Moreover, we have determined several high-resolution crystal structures of the pathogenic MRSA enzyme in complex with representative inhibitors, placing us in a strong position to further develop these new antibiotics. The efforts to develop an efficacious candidate compound will evolve through three specific aims. In the first Aim, we will complete an initial analog series and select two lead compounds to move forward into animal studies in the second specific aim. These studies will determine key pharmacokinetic parameters (bioavailability, half-life) for our compounds as well as determine efficacy in an animal model of infection. In the third specific aim, we will explore new, structure-based designs to enhance the selectivity of our compounds for the pathogenic forms of DHFR over the human homolog. Completion of these studies will position us to attract outside investors and partners to progress our compounds for an IND application. PUBLIC HEALTH RELEVANCE: Despite decades of work on the discovery of antibiotics, the continued emergence of resistance organisms threatens to render many of our best antibiotics obsolete. We are working to develop new agents against that function as effective monotherapies against the methicillin-resistant strain of Staphylococcus aureus (MRSA) and Streptococcus pyogenes. Specifically we are targeting the essential enzyme dihydrofolate reductase (DHFR) that is required by the bacteria to synthesize key components needed to replicate its genetic material.